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Background: The ability to undertake molecular analysis to inform on prognosis and predictors of response to therapy is limited by accessibility of tissue. Measurement of total circulating free DNA (cfDNA) or circulating tumor DNA (ctDNA) in peripheral blood may allow easier access to tumor material and help to predict clinical outcomes.Methods: A systematic review of electronic databases identified publications exploring the association between cfDNA or ctDNA and overall survival (OS) in solid tumors. HRs for OS were extracted from multivariable analyses and included in a meta-analysis. Pooled HRs were computed and weighted using generic inverse variance and random-effect modeling. For studies not reporting multivariable analyses, univariable ORs were estimated from Kaplan–Meier curves for OS at 1 and 3 years.Results: Thirty-nine studies comprising 4,052 patients were included in the analysis. Detection of ctDNA was associated with a significantly worse OS in multivariable analyses [HR, 2.70; 95% confidence interval (CI), 2.02–3.61; P < 0.001). Similar results were observed in the univariable analyses at 3 and 1 year (OR, 4.83; 95% CI, 3.20–7.28; P < 0.001).There was also a statistically significant association between high total cfDNA and worse OS for studies reporting multivariable and univariate data at 3 years (HR, 1.91; 95% CI, 1.59–2.29; P < 0.001 and OR, 2.82; 95% CI, 1.93–4.13; P < 0.001, respectively).Conclusions: High levels of total cfDNA and presence of ctDNA are associated with worse survival in solid tumors.Impact: Circulating DNA is associated with worse outcome in solid tumors. Cancer Epidemiol Biomarkers Prev; 25(2); 399–406. ©2015 AACR.

Supplementary Figure 1: Forest plot showing the pooled odds ratio (OR) for overall survival at 1 year for circulating tumor DNA. Supplementary Figure 2: Forest plot showing the pooled odds ratio (OR) for overall survival at 1 year for total circulating DNA. Supplementary Figure 3: Forest plot showing A: pooled hazard ratio (HR) for overall survival for total tumor circulating DNA based on method of collection (plasma versus serum) and B: pooled hazard ratio (HR) for overall survival for t total circulating DNA based on method of collection (plasma versus serum).

Detailed description of the included studies.

It has been proposed that cell-free nucleic acids in the plasma participate in tumorigenesis and the development of metastases via transfection-like uptake of such nucleic acids by susceptible cells. This putative phenomenon is tentatively referred to as “genometastasis.” In the present study, we examined the effects on cultured cells of plasma from healthy individuals and from patients with colon cancer. Cultures of NIH-3T3 cells and human adipose-derived stem cells (hASC) were supplemented with samples of plasma from patients with K-ras–mutated colorectal tumors or from healthy subjects using two different protocols: direct addition of plasma to cultures in standard plates and addition in the absence of contact between plasma and cells, which were separated by a membrane with 0.4-μm pores. In plasma-treated hASCs, no K-ras–mutated sequences were detected by real-time PCR. In contrast, in most cultures of plasma-treated NIH-3T3 cells (murine cells), the transfer of human DNA occurred, as verified by the detection of human K-ras sequences, p53 sequences, and β-globin–encoding sequences. Moreover, NIH-3T3 cells that had been cultured with plasma from patients with colon cancer were oncogenically transformed, as shown by the development of carcinomas in nonobese diabetic–severe combined immunodeficient mice after the injection of such cells. Microscopic analysis of membranes that had separated plasma from cultured cells confirmed the complete absence of cells in the plasma. We only observed noncell particles, having diameters of

Supplementary Methods from Cell-Free Nucleic Acids Circulating in the Plasma of Colorectal Cancer Patients Induce the Oncogenic Transformation of Susceptible Cultured Cells

Endoscopic ultrasound (EUS) procedures are becoming more frequent nowadays and novel techniques are on the rise. These procedures require high technical experience and complex endoscopic skills. The goal of this study was to develop a new minimally invasive animal model of bile duct dilatation in the pig, in order to offer a new tool for endoscopic and surgical therapy training and to test new therapeutic strategies. Twenty-five female pigs underwent laparoscopic surgery in order to perform a common hepatic duct ligation. A pre- and postoperative biochemical analyses were performed: glucose, albumin, total bilirubin (TBil), gamma glutamyl transferase (GGT), alkaline phosphatase, and alanine aminotransferase were measured. Surgical time and intra- and postoperative complications were registered. Five to six days after surgery, an EUS was performed to measure intrahepatic duct size (mm). Distance from the bile duct to the EUS transductor was also recorded (mm). T-student for quantitative variables was applied. Statistical significance was defined as p value ≤ 0.05. The mean surgical time was 29.5 ± 14.9 min. In five pigs (20%), some mild intraoperative problems occurred. A severe postoperative complication occurred in one animal (4%). No postoperative mortality was registered. Postoperative serum analyses showed an increase in total bilirubin (p = 0.005) and gamma glutamyl transferase levels (p = 0.001). Postoperative EUS showed dilatation of the intrahepatic bile duct in 76% of pigs, with a mean diameter of 9.6 ± 3.6 mm (distance from the gastric wall of 17.0 ± 6.4 mm). The surgical procedure described here is a safe technique to induce dilatation of the intrahepatic bile ducts in the pig, with a minimally invasive approach and a high efficacy rate. This animal model might be useful for EUS techniques training and for evaluating new therapeutic approaches.

Elevated mRNA expression of human telomerase reverse transcriptase (hTERT mRNA) is common in many types of tumors, participating in tumor growth and progression. Such expression has not been sufficiently examined in renal cancer. The goal of the present study was to quantify it and analyze its possible clinical value in the management of this pathology.The study included 111 patients who underwent surgery for renal cell carcinoma (RCC) between 2015 and 2017. Tumor samples were taken from all patients and, in 94 of them, healthy renal tissue adjacent to the tumor was also sampled. The 2 types of tissue were histologically confirmed, after which mRNA was extracted. Using real-time quantitative PCR, the expression of hTERT and glyceraldehyde-3-phosphate dehydrogenase (as endogenous control) were indirectly quantified using the crossing point (CP), which is inversely correlated with the number of sample replicates yielding positive results. These values were correlated with patient socio-demographic variables and clinical-pathological factors of the RCC.The majority of patients were males, with an average age of 60.5 years (SD: 14.02). Most tumors (69.4%) were clear cell carcinomas. The most frequent stages were pT2 or lower (73%), while 5% were pN1 and 12% pM1. The majority of tumors (58%) were Fuhrman grades 1 or 2 (low grade). All samples of tumor and nontumor tissue expressed glyceraldehyde-3-phosphate dehydrogenase mRNA, with the CP in the tumor sample significantly lower than in the nontumor tissue (P0.001). The expression of hTERT mRNA was detected in 68% of tumor tissues and significantly correlated with histopathology: 100% in sarcomatoid RCC and 77.9% in clear cell carcinomas (P0.0001). The CP was lower in pN1 (P = 0.018), pM1 (P = 0.046), and TNM IV stages (P = 0,041). A greater number of hTERT mRNA replicas were detected in M1 patients (P = 0.0005) and TNM IV stage (P = 0.017). There was no correlation of hTERT mRNA expression with Fuhrman grade.The quantitation of hTERT mRNA expression in RCC might be useful as a complementary diagnostic tool as well as for assessing aggressiveness of the tumor.

Background Traumatic brain injury (TBI) is one of the most frequent and severe neurological diseases. In the last few decades, significant advances have been made in TBI pathophysiology and monitoring, however new treatments have not emerged. Although the central nervous system (CNS) has been historically defined as an immunologically privileged organ, recent studies show the increasingly predominant role of inflammatory and apoptotic phenomena in the pathogenesis of TBI. Inflammatory response mediators can be eliminated with continuous renal replacement therapies (CRRT). Our aim was to investigate whether hemofiltration protects the brain after head trauma in an experimental study in animals. Methods and results A model of TBI and CVVH was performed in anesthetized New Zealand white rabbits without acute renal failure. The experimental group TBI ( +)-CVVH ( +) was compared with a TBI ( +)-CVVH (−) and a TBI (−)-CVVH ( +) control groups. Rabbits were assessed immediately (NES1) and 24 h hours after (NES2) TBI and/or CVVH using a functional Neurological Evaluation Score (NES) and histology of the brains after sacrifice. There was evidence to support a difference of NES1 comparing with the TBI (−)-CVVH ( +), but not with TBI ( +)-CVVH (−) with only 15% of the rabbits treated with CVVH and TBI showing a favorable neurological course. The final neurological outcome (mortality at 24 h) was 0%, 22% and 53% in the TBI(−) + CVVH( +), TBI( +)-CVVH(−) and TBI( +)-CVVH( +) groups respectively. The use of hemofiltration before or after TBI did not make a difference in regards the outcome of the rabbits. There was evidence in the histology to support an increase of mild ischemia, hemorrhage and edema in the experimental group compared with the other two groups. Conclusions CVVH in rabbits without renal failure used with the intention to protect the brain may worsen the prognosis in TBI.

Differential presence of exons (DPE) is a method of interpretation of exome sequencing, which has been proposed to design a predictive algorithm with clinical value in patients with colorectal cancer (CRC). The goal of the present study was to examine the reproducibility in a rat model of metastatic colon cancer. DHD/K12-TRb cells were injected in syngenic immunocompetent BD-IX rats. Cells were from two stocks with low and normal metastatic potential, and injected into two separate groups of rats. Five to ten weeks after injection, blood samples were taken prior euthanasia and whole exome sequencing performed. Through DPE analysis, we identified a set of exons whose differential presence in plasma allowed us to compare both groups of tumor-bearing animals. A verification test was performed to confirm that the algorithm was able to classify extracted samples into their corresponding groups of origin. The highest mean probability was 0.8954. In conclusion, the DPE analysis in tumor-bearing animals was able to discriminate between different disease status, which fully supports previous results in CRC patients., This studywas funded by two grants from"Instituto de Salud Carlos III", Spain (FIS; refs. PS09/01815 and PI13/01924)

Most colorectal cancer (CRC)-related deaths are due to liver metastases. PKCζ is a tumor suppressor in CRC with reduced expression in metastasis. Given the importance of microRNAs (miRNAs) in regulating cellular plasticity, we performed an unbiased screening and identified the miR-200 family as the most relevant miRNAs downregulated by PKCζ deficiency. The regulation of the intracellular levels of miR-200 by PKCζ is post-transcriptional and involves their secretion in extracellular vesicles. Here, we identified ADAR2 as a direct substrate of PKCζ in CRC cells. Phosphorylation of ADAR2 regulates its editing activity, which is required to maintain miR-200 steady-state levels, suggesting that the PKCζ/ADAR2 axis regulates miR-200 secretion through RNA editing. Loss of this axis results in epithelial-to-mesenchymal transition (EMT) and increased liver metastases, which can be inhibited in vivo by blocking miR-200 release. Therefore, the PKCζ/ADAR2 axis is a critical regulator of CRC metastases through modulation of miR-200 levels. Shelton et al. demonstrate that the loss of the tumor suppressor PKCζ in colorectal cancer cells results in the downregulation of miR-200, leading to increased epithelial-to-mesenchymal transition, cell invasion, and liver metastasis. This is mediated by an increase in miR-200 secretion through the inactivation of the RNA editing enzyme ADAR2, identifying a key vulnerability in liver metastasis, Research was supported by grants from the NIH (R01DK108743, R01CA172025, and R01CA207177 to J.M.; R01CA192642 and R01CA218254 to M.T.D.-M.)

BACKGROUND The use of plasma as a "liquid biopsy" has gained increasing attention. The purpose of the present study was to evaluate the diagnostic and prognostic utility of the perioperative detection and quantitation of mRNAs encoding human telomerase reverse transcriptase (hTERT) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in plasma from patients with cancer of the larynx or hypopharynx. METHODS We recruited 47 patients with laryngeal cancer and 2 patients with hypopharyngeal cancer, plus 27 healthy subjects. A blood sample was taken from each patient before and after surgical resection of the tumor. We quantified hTERT mRNA and GAPDH mRNA in plasma by real-time polymerase chain reaction (PCR). RESULTS Detection of hTERT mRNA before surgery had diagnostic value (sensitivity, 22%; specificity, 100%). Detection was more frequent in patients with supraglottic tumors than glottic tumors (p = .02) and was related to subsequent recurrence (p = .02). Preoperative levels of hTERT mRNA in plasma were higher in patients with subsequent recurrence (p = .046) and/or metastases (p = .047). The disease-free survival (DFS) and overall survival (OS) of patients with plasma samples positive for hTERT mRNA was poorer than that of patients with negative samples. Mean levels of plasma GAPDH mRNA in untreated patients were higher than in healthy subjects (p < .001). CONCLUSION Detection and quantitation of hTERT and GAPDH mRNA in patients' plasma might be clinically significant in cases of laryngeal and hypopharyngeal cancer. © 2017 Wiley Periodicals, Inc. Head Neck 39: 647-655, 2017.

The abdomen is the second most common source of sepsis and secondary peritonitis, which likely lead to death. In the present study, we hypothesized that instillation of local anesthetics into the peritoneum might mitigate the systemic inflammatory response syndrome (SIRS) in the open abdomen when combined with negative-pressure therapy (NPT) to treat severe peritonitis. We performed a study in 21 pigs applying a model of sepsis based on ischemia/reperfusion and fecal spread into the peritoneum. The pigs were randomized into three groups, and treated for 6 h as follows: Group A: temporary abdominal closure with ABTHERA™ Open Abdomen Negative-Pressure Therapy; Group B: temporary abdominal closure with ABTHERA™ Open Abdomen Negative-Pressure Therapy plus abdominal instillation with physiological saline solution (PSS); and Group C: temporary abdominal closure with ABTHERA™ Open Abdomen Negative-Pressure Therapy plus peritoneal instillation with a solution of ropivacaine in PPS. A comparison between the three groups revealed no statistically significant difference for any of the parameters registered (p > 0.05), i.e., intra-abdominal pressure, blood pressure, heart rate, O2 saturation, diuresis, body temperature, and blood levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), and c-reactive protein (CRP). In addition, histological studies of the liver, ileum, kidney and lung showed no difference between groups. The use of abdominal instillation (with or without ropivacaine) did not change the effect of 6 h of NPT after sepsis in animals with open abdomen. The absence of adverse effects suggests that longer treatments should be tested.

Increasingly effective therapies targeting the androgen receptor have paradoxically promoted the incidence of neuroendocrine prostate cancer (NEPC), the most lethal subtype of castration-resistant prostate cancer (PCa), for which there is no effective therapy. Here we report that protein kinase C (PKC)λ/ι is downregulated in de novo and during therapy-induced NEPC, which results in the upregulation of serine biosynthesis through an mTORC1/ATF4-driven pathway. This metabolic reprogramming supports cell proliferation and increases intracellular S-adenosyl methionine (SAM) levels to feed epigenetic changes that favor the development of NEPC characteristics. Altogether, we have uncovered a metabolic vulnerability triggered by PKCλ/ι deficiency in NEPC, which offers potentially actionable targets to prevent therapy resistance in PCa.

Highlights • Plasma levels of GAPDH cfDNA might be useful as complementary methods for diagnosis and prognosis of renal cell carcinoma. • Pre and postoperative levels of GAPDH cfDNA were independently associated with OS in renal cell carcinoma. • hTERT cfRNA was not a marker useful for diagnosis nor prognosis of renal cell cancer., Introduction The current challenge on renal cell carcinoma (RCC) is to finding a non-invasive biomarker for improving their diagnostic and therapeutic management. In the present study, we analyzed the clinical value of plasma levels of cell-free DNA (cfDNA) and RNA (cfRNA) of two genes: glyceraldehyde 3-phosphate-dehydrogenase (GAPDH) and human telomerase reverse transcriptase (hTERT). Materials and methods We recruited 82 patients with RCC, and 20 healthy subjects. Using RT-PCR techniques, plasma levels of cfDNA and cfRNA from hTERT and GAPDH genes were quantified pre- and post-operatively, and one year after surgery. Relationships between such plasma levels and clinicopathological features and evolution of disease were analyzed. Results Levels of GAPDH cfDNA and cfRNA were significantly higher in patients than in healthy subjects. hTERT cfDNA was detected in plasma from 35% of RCC patients and in none healthy subject. At diagnosis, plasma levels of GAPDH cfDNA were higher in advanced pT and TNM stages, and hTERT cfDNA in patients with 3–4 Fuhrman grade and affected lymph nodes. Levels of cfNAs were not related to the presence of metastasis. Following nephrectomy, GAPDH cfDNA levels dropped, and patients with higher levels before and after nephrectomy, showed lower overall survival (OS). However, Cox's multivariate model did not prove any association of the cfNA levels with progression. Conclusion Plasma levels of cfDNA from GADPH and hTERT genes were correlated to tumor diagnosis and progression and, thus, such analyses might help to diagnosis and prognosis of RCC patients.

Background: To examine the usefulness of green fluorescent protein (GFP) mice for studying the interactions between normal cells and tumor cells in a host, we used a melanoma model in such “green” mice [C57BL/6-Tg(CAG-EGFP)1Osb mice]. Mice were given a subcutaneous injection of B16-F10 cells, and the resultant primary tumors were removed. Then cells from individual tumors were cultured. Results: The proportion of EFGP+ cells was determined by fluorescence-activated cell sorting (FACS) and was 6.8% ± 3.2% (mean ± s.d.) on day 1 of culture, 0.6% ± 0.3% on day 2, and 0.02% ± 0.01% at day 7. In all cases, isolated cells grew at a constant rate, but fluorescence decreased over time and became undetectable on day 14. Cells were tested using PCR for the presence of an EGFP-specific sequence, and results were negative in all cases, thus indicating that the cells did not harbor the host's reporter gene. Cells were also tested for the presence of EGFP mRNA, which was consistently detected for 22 days after the start of culture. The tumorogenicity of the cultured cells was confirmed in GFP mice injected with cells from a selection of cultures. Conclusions: In a melanoma model in GFP mice, the detection of “green” cells in tumors was not equivalent to the detection of host-derived cells. Such “masking” was caused by a transient, but lasting, transfer of EGFP mRNA from the host's normal cells to tumor cells. Thus, an analysis of tumors postmortem by techniques that yield only a single snapshot can lead to incorrect interpretations and erroneous conclusions.How to cite: Garcia-Olmo D, Picazo MG, García-Olmo D. Melanoma transplants in “green” mice: fluorescent cells in tumors are not equivalent to host-derived cells. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.04.007. Keywords: Cancer models, Development of tumors, EGFP, GFP mice, Green-fluorescent cells, Melanoma, Mouse, Oncogenic transformation

The E1a gene from adenovirus has become a major tool in cancer research. Since the discovery of E1a, it has been proposed to be an oncogene, becoming a key element in the model of cooperation between oncogenes. However, E1a's in vivo behaviour is consistent with a tumour suppressor gene, due to the block/delay observed in different xenograft models. To clarify this interesting controversy, we have evaluated the effect of the E1a 13s isoform from adenovirus 5 in vivo. Initially, a conventional xenograft approach was performed using previously unreported HCT116 and B16-F10 cells, showing a clear anti-tumour effect regardless of the mouse's immunological background (immunosuppressed/immunocompetent). Next, we engineered a transgenic mouse model in which inducible E1a 13s expression was under the control of cytokeratin 5 to avoid side effects during embryonic development. Our results show that E1a is able to block chemical skin carcinogenesis, showing an anti-tumour effect. The present report demonstrates the in vivo anti-tumour effect of E1a, showing that the in vitro oncogenic role of E1a cannot be extrapolated in vivo, supporting its future use in gene therapy approaches., This work was supported by grants from Fundación Leticia Castillejo Castillo and MINECO (SAF 2015 64215-R) and from JCCM (PPII10-0141-0404) to RSP. F. Cimas was supported by Fundación Leticia Castillejo Castillo and Cátedra Enresa. RSP research institution received support from the European Community through the regional development funding programme (FEDER).

KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker., This study was funded by a grant from “Fondo de Investigaciones Sanitarias-FEDER”, Ministry of Health, Spain (FIS; PI13/01924) and the Spanish Ministry of Health and Consumer Affairs (via a cooperative network-FEDER [TerCel RD12- 0019-0035])

[Background]: The ability to undertake molecular analysis to inform on prognosis and predictors of response to therapy is limited by accessibility of tissue. Measurement of total circulating free DNA (cfDNA) or circulating tumor DNA (ctDNA) in peripheral blood may allow easier access to tumor material and help to predict clinical outcomes. [Methods]: A systematic review of electronic databases identified publications exploring the association between cfDNA or ctDNA and overall survival (OS) in solid tumors. HRs for OS were extracted from multivariable analyses and included in a meta-analysis. Pooled HRs were computed and weighted using generic inverse variance and random-effect modeling. For studies not reporting multivariable analyses, univariable ORs were estimated from Kaplan-Meier curves for OS at 1 and 3 years. [Results]: Thirty-nine studies comprising 4,052 patients were included in the analysis. Detection of ctDNA was associated with a significantly worse OS in multivariable analyses [HR, 2.70; 95% confidence interval (CI), 2.02-3.61; P < 0.001). Similar results were observed in the univariable analyses at 3 and 1 year (OR, 4.83; 95% CI, 3.20-7.28; P < 0.001).There was also a statistically significant association between high total cfDNA and worse OS for studies reporting multivariable and univariate data at 3 years (HR, 1.91; 95% CI, 1.59-2.29; P < 0.001 and OR, 2.82; 95% CI, 1.93-4.13; P < 0.001, respectively). [Conclusions]: High levels of total cfDNA and presence of ctDNA are associated with worse survival in solid tumors. [Impact]: Circulating DNA is associated with worse outcome in solid tumors., This work was supported by grants from CRIS cancer foundation (to A. Ocaña and A. Pandiella).

The use of plasma as a "liquid biopsy" has gained increasing attention. The purpose of the present study was to evaluate the diagnostic and prognostic utility of the perioperative detection and quantitation of mRNAs encoding human telomerase reverse transcriptase (hTERT) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in plasma from patients with cancer of the larynx or hypopharynx.We recruited 47 patients with laryngeal cancer and 2 patients with hypopharyngeal cancer, plus 27 healthy subjects. A blood sample was taken from each patient before and after surgical resection of the tumor. We quantified hTERT mRNA and GAPDH mRNA in plasma by real-time polymerase chain reaction (PCR).Detection of hTERT mRNA before surgery had diagnostic value (sensitivity, 22%; specificity, 100%). Detection was more frequent in patients with supraglottic tumors than glottic tumors (p = .02) and was related to subsequent recurrence (p = .02). Preoperative levels of hTERT mRNA in plasma were higher in patients with subsequent recurrence (p = .046) and/or metastases (p = .047). The disease-free survival (DFS) and overall survival (OS) of patients with plasma samples positive for hTERT mRNA was poorer than that of patients with negative samples. Mean levels of plasma GAPDH mRNA in untreated patients were higher than in healthy subjects (p.001).Detection and quantitation of hTERT and GAPDH mRNA in patients' plasma might be clinically significant in cases of laryngeal and hypopharyngeal cancer. © 2017 Wiley Periodicals, Inc. Head Neck 39: 647-655, 2017.